1. Field of Invention
The invention relates to systems and methods for detecting light using a sensor. More specifically, the systems and methods of the invention relate to an imaging detector that includes an array of sensors.
2. Description of Related Art
Fluorescence illumination and observation is a rapidly expanding microscopy technique employed today. This microscopy technique may be used in both the medical and biological sciences. Because the microscopy technique is rapidly expanding, sophisticated microscopes and numerous fluorescence accessories have been developed. For example, Epi-fluorescence, or incident light fluorescence, is used in many applications. The technique of fluorescence microscopy has become an essential tool in biology and the biomedical sciences, as well as in materials science due to attributes that are not readily available in other contrast modes with traditional optical microscopy. The application of an array of fluorochromes has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity among non-fluorescing material. In fact, the fluorescence microscope is capable of revealing the presence of a single molecule. Through the use of multiple fluorescence labeling, different probes can simultaneously identify several target molecules.
Fluorescence microscopy includes a process in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism. The generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed photoluminescence, which may be divided into two categories: fluorescence and phosphorescence. Each process depends upon the electronic configuration of the excited state and the emission pathway. The absorption and subsequent re-radiation of light by organic and inorganic specimens is generally the result of the fluorescence or phosphorescence. The fluorescence process uses the ability of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after a brief interval. The process of phosphorescence occurs in a manner similar to fluorescence, but with a much longer excited state duration. The emission of light through the fluorescence process is nearly simultaneous with the absorption of the excitation light. When emission persists longer after the excitation light has been extinguished, the phenomenon is referred to as phosphorescence.
The modern fluorescence microscope may combine the power of high performance optical components with computerized control of the instrument and digital image acquisition to achieve a level of sophistication that far exceeds that of simple observation by the human eye. The fluorescence microscopy generally depends on electronic imaging to rapidly acquire information at low light levels or at visually undetectable wavelengths.
In contrast to other modes of optical microscopy that are based on macroscopic specimen features (such as phase gradients, light absorption, and birefringence), fluorescence microscopy may image the distribution of a single molecular species based solely on the properties of fluorescence emission. Thus, using fluorescence microscopy, a precise location of intracellular components labeled with specific fluorophores may be monitored, as well as their associated diffusion coefficients, transport characteristics, and interactions with other biomolecules. In addition, the response in fluorescence to localized environmental variables enables the investigation of pH, viscosity, refractive index, ionic concentrations, membrane potential, and solvent polarity in living cells and tissues.
One benefit of fluorescence microscopy is its ability to detect fluorescent objects that are sometimes faintly visible or even very bright relative to the dark (often black) background. In order to achieve this benefit, image brightness and resolution must be maximized by ensuring that the object or sample is supplied with sufficient light energy for excitation at the appropriate wavelength for each chromophore attached to the specimen. Moreover, a selection of a proper filter will maximize the amount of emitted fluorescence directed to the sensor.
Two examples of commonly used light detectors used in fluorescence microscopy are the photomultiplier tube (PMT) and the photodiode. Both devices employ a photosensitive surface that captures incident photons and generates electronic charges that are sensed and amplified. PMTs are commonly used in confocal microscopes and high-end automatic exposure bodies for film cameras as well as in spectrometers. These devices respond when photons impinge on a photocathode and liberate electrons that are accelerated toward an electron multiplier composed of a series of curved plates (known as dynodes). Conventional methods that use a single detector such as a single PMT to detect light, or illuminate the object broadly and detect the light using a pixel array may not properly detect the emitted fluorescence light. For example, the detection using these methods may be unsatisfactory and relatively slow because a single detector is used in the device with the PMT. Moreover, a weak signal may result when broadly scanning the pixel array thus making detection of the light even more difficult.
Silicon photodiodes may also be used to respond rapidly to light by the generation of a current. Uniformity of the photosensitive surface is excellent and the dynamic range and response speed of these devices are among the highest of any light detector. However, conventional arrangements using the silicon photodiodes have a relatively flat response over the entire visible spectrum. Moreover, conventional arrangements of silicon diodes as sensors may also produce a considerable amount of noise, (much of it thermal) resulting in relatively poor signal-to-noise under photon-limited conditions.
Fluorescence microscopes may be used to irradiate the specimen with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light. Ideally, only the emission light should reach the detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark (or black) background. However, conventional devices do not properly detect the emitted fluorescence light in this manner using conventional sensor arrangements.
Some conventional systems perform detection by putting a lens in front of the fixed point and a detector behind the lens. The detector collects all of the light that was emitted through the lens. However, in these systems, not all of the light is collected and detected, which poses a particular problem if the angular distribution of the emission is large. Moreover, the time required to accurately detect using this system is excessive.
When the detection device uses the PMT imager scanned across the plate, or a charge-coupled device (CCD) to image a line, the time necessary for imaging is excessive. In the case of a CCD, the detector is optically coupled to the capillary array by way of the capillaries in the array being optically coupled to the linearly aligned pixels. However, this method is disadvantageous because the illumination at any given cell is generally very-weak, and the optical coupling between the object and detector may be unsatisfactory when a large field of view is needed.
If the conventional fluorescence detection device includes PMTs, CCDs, optical filters, lenses and lasers, the detection system may tend to be bulky resulting in problems that can arise because of the size of the detections systems. Fluorescent detection processes are very sensitive, especially when they are combined with laser excitation. However, the detection systems of the prior techniques pose many problems in the efficiency of sequencing and imaging. For example, some fixed end detection systems require up to eight hours in order to detect one sample. Further, by using a prior art detector, all of the possible data may not be collected. Another reason that the prior art detection systems are not efficient is that these systems typically only detect one band at a time, e.g. the band that has reached the end of the separation apparatus in fixed end detection.